Modified-affinity streptavidin

ABSTRACT

Streptavidin tetramers have at least one monomer containing an amino acid modification that produces a reduced binding affinity for biotin, a modified off-rate, a modified on-rate, or an altered binding enthalpy. Polynucleotides encoding the modified monomers are also provided. The modified streptavidin and chimeric streptavidin molecules are useful in methods of bioseparations and cell sorting, imaging, drug delivery, and diagnostics.

GOVERNMENT SUPPORT

Certain embodiments of the invention described herein were made in thecourse of work supported by the National Science Foundation. Therefore,the U.S. Government has certain rights in this invention.

This application is a continuation of Ser. No. 08/387,055, filed Feb. 9,1995.

BACKGROUND OF THE INVENTION

Streptavidin, a protein produced by Streptomyces avidinii, forms a verystrong and specific non-covalent complex with the water soluble vitaminbiotin. Streptavidin is a tetrameric protein that binds biotin with anaffinity that is amongst the highest displayed for non-covalentinteractions between a ligand and protein, with an association constant(Ka) estimated to be in the range of 10¹³ M⁻¹ to 10¹⁵ M⁻¹. This bindingaffinity is strong enough to be essentially irreversible under normalphysiological solution conditions, and provides the basis forstreptavidin and biotin's usefulness in a wide variety of clinical andindustrial applications. See, Green, Adv. Prot. Chem. 29: 85-143 (1975).

Both streptavidin and the homologous protein avidin, which shares itshigh affinity for biotin, have been studied as paradigms of strongligand-protein interactions. The X-ray crystal structures ofstreptavidin and avidin, both in their apo and holo forms, have beendescribed. The sequences of both have also been reported, as have theconstruction of several streptavidin fusion proteins (Sano and Cantor,Biochem. Biophys. Res. Commun. 176: 571-577 (1991); U.S. Pat. No.4,839,293). The structure-function origins of the unusually highaffinity, however, have yet to be elucidated.

The streptavidin molecule displays a number of the common recognitionmotifs that have been identified for protein-ligand bindinginteractions. These include van der Waals dispersive attractions, whichare largely mediated by the aromatic side chains of tryptophan residues,hydrogen bonding networks mediated by donor/acceptor side chains, anddisorder-to-order transitions mediated by the ordering of surfacepolypeptide loops upon ligand binding.

Miyamoto and Kollman have reported a computational study that emphasizesthe importance of hydrophobic/van der Waals dispersive attractionbetween ligand and protein. Miyamoto and Kollman, Proteins 16: 226-245(1993) and Proc. Natl. Acad. Sci. USA 90: 8402-8406 (1993). Thesestudies suggest that hydrophobic/van der Waals interactions contribute.sup.˜ 18 kcal/mol to the absolute free energy of binding, while theelectrostatic energy term (which includes hydrogen bonding interactions)contributes only .sup.˜ 3 kcal/mol.

In addition to the extremely high binding affinity, the usefulness ofstreptavidin also arises from the unique architectural properties of theprotein. Streptavidin is a tetramer of four identical subunits, witheach subunit contributing a binding site for biotin. Because thetetramer has approximate two-fold symmetry, the binding sites arepositioned in pairs on opposite sides of the molecule, making theprotein an efficient molecular adaptor. This structural feature, alongwith the high affinity of streptavidin for biotin, has made the proteinan important component in many technologies.

While the streptavidin tetramer displays nearly ideal 222 point groupsymmetry, there are two distinct protein-protein interfaces within thetetramer. Hendrickson et al., Proc. Natl. Acad. Sci. USA 86: 2190-2194(1989); Weber et al., Science 243:85-88 (1989). The first interface liesbetween two monomers that are related by the two-fold symmetry axis, andis defined by an extensive overlap of β-barrel surfaces withcomplementary curvatures. This interface is characterized by a number ofvan der Waals, hydrogen bonding, and salt-bridge interactions. The closeassociation of subunits at this interface defines the streptavidindiner, with biotin binding sites related by the two-fold symmetry axis.The second tetramer interface defines the surface between pairs of theseclosely associated dimers (streptavidin is well described as a "dimer ofdimers"). The dimer/dimer interface is characterized by a very loose"waistline" with minimal bonding interactions mediated largely by theC-terminal β-strand 8 of the monomers. Thus, the dimer interface isstructurally extensive while the dimer/dimer interface is structurallyminimal. Despite the apparent lack of strong bonding interactions at thedimer/dimer interface, the streptavidin tetramer is exceedingly stablein both the biotin-free and biotin-bound states. The tetramer does notdissociate into smaller subunits in either 6 M urea or 6 M guanidiniumhydrochloride. Kurzban et al., J. Biol. Chem. 266: 14470-14477 (1991).The biotin binding site is defined by a plurality of amino acidresidues, as shown, for example, by FIG. 3 of Weber et al.

Streptavidin and avidin are key components in four technological areasof great significance: 1) bioseparations/cell sorting; 2) imaging; 3)drug delivery; and 4) diagnostics (Wilchek and Bayer, in Meths. Enzymol.184: 5-45 (1990)). In the separations area, these proteins have beenused extensively in important cell sorting applications, where forexample they are used to remove contaminating cells from hematopoieticstem cells prior to marrow transplantation. Berenson et al., Prog. Clin.Biol. Res. 377: 449-459 (1992). They have found similar wide use incancer diagnostics, where they are used extensively in both research andclinical settings to test for the presence of various tumor specificbiomarkers.

The imaging and drug delivery applications of streptavidin/avidin andbiotin arise from the capability for simultaneous targeting and deliveryof imaging agents or therapeutics to tumor cells. There is particularlysignificant emerging interest in the use of streptavidin/avidin fortargeted delivery of imaging agents and therapeutics in vivo.Streptavidin/avidin has been used to deliver drugs, toxins and imagingagents to targeted cells both in vitro and in vivo. See, e.g., Meyer etal., Exp. Hematol. 19: 710-713 (1991). In these systems, streptavidinplays the crucial role of molecular adaptor between an antibody thatserves as the targeting component, and a biotinylated therapeutic orimaging agent. With some strategies, cells are pre-targeted with theantibody-streptavidin conjugate, with subsequent delivery of thebiotinylated agent. In other applications, a biotinylated antibody isfirst used to pre-target cells, with subsequent delivery of thestreptavidin-biotinylated agent conjugate. A three-step delivery is alsopossible, using biotinylated antibody followed by streptavidin and thenthe biotinylated agent.

While streptavidin and avidin are incredibly useful molecules, they haveimportant limitations, such as the inflexibility of four identicalsubunits having binding sites with extremely high affinity. Further, ithas not been feasible to control the distribution of the subunits withinthe tetramer if the degeneracy of the subunits is removed (e.g.,subunits with different affinities, subunits labeled with differentimaging agents, subunits labeled with different drugs).

What is needed in the art is the ability to tailor the functionalproperties of individual subunits, and their geometrical distributionwithin the tetramer. This can be accomplished by manipulating importantstreptavidin structure-function relationships. A library of streptavidinmutants spanning a range of affinities and off- and on-rates for biotinand its derivatives would improve upon existing biotechnologicalapplications for this already widely used system and open it toimportant new uses. Similarly, the ability to precisely define thesubunit components and geometry will dramatically improve existingapplications and provide new tools for cell separations, imaging,therapeutics and a variety of other technologies. Quite surprisingly,the present invention fulfills these and other related needs.

SUMMARY OF THE INVENTION

The present invention provides a streptavidin tetramer where at leastone monomer of the tetramer has an amino acid modification that producesa reduced binding affinity for biotin, a modified off-rate, a modifiedon-rate, and/or an altered binding enthalpy. The resulting bindingaffinity of the tetramer for biotin is less than approximately 1×10¹³M⁻¹. Typically, at least one monomer of the streptavidin tetramer willhave an amino acid modification that results in the reduced bindingaffinity for biotin, sometimes at least two or three monomers, and insome embodiments all four monomers which comprise the tetramer have anamino acid modification that results in the reduced binding affinity forbiotin. In a preferred embodiment, the amino acid modification is asubstitution of a tryptophan residue in a biotin binding site, such asthe tryptophan residue at amino acid position 79, 92, 108 or 120. Thetryptophan residue of at least two of the foregoing amino acid positionscan be substituted or deleted. Conveniently, any of the amino acidsother than tryptophan lowers the binding affinity and results in afaster off-rate; phenylalanine or alanine are preferred substitutionsfor the tryptophan residue in the biotin binding site described in theillustrative examples described herein.

The invention also provides an isolated polynucleotide molecule whichencodes streptavidin having an amino acid modification that results in areduced binding affinity of streptavidin for biotin. In representativeembodiments the polynucleotide molecule encodes an amino acidsubstitution or deletion of a tryptophan residue in a biotin bindingsite, such as a tryptophan residue at amino acid position 79, 92, 108 or120, e.g., by substituting a phenylalanine or alanine residue or otheramino acid residue. The isolated polynucleotide molecule may encodesubstitutions of at least one of said amino acid positions, sometimestwo or more.

In other aspects the invention provides a method for producing astreptavidin tetramer with at least one monomer thereof having acharacteristic not found in a native streptavidin monomer subunit whichcharacteristic affects affinity but not specificity for biotin. Themethod comprises producing altered streptavidin tetramer having thecharacteristic, separating the altered streptavidin into monomer and/ordiner subunits, e.g., by guanidium thiocyanate refolding; and mixing thestreptavidin monomer and/or dimer subunits with streptavidin monomer ordiner subunits which do not have the characteristic, thereby producing astreptavidin tetramer having at least one monomer thereof with saidcharacteristic. The method can also be used to assemble chimerictetramers where at least one of the monomers but less than all containsa label, drug, toxin, targeting molecule, metal, or an amino acidmodification that results in a reduced binding affinity of streptavidinfor biotin. For example, the streptavidin monomer may contain at leastone mutation in the amino acid sequence thereof which is situated at thedimer/dimer interface, e.g., a disulfide bond can be engineered toconnect specific subunits and define the stoichiometry of thedissociated species.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates mixing of subunits with different labels to formchimeric tetramers by using the fluorescence resonance energy transferof CPI-and FITC-labeled WT streptavidin after guanidine thiocyanateinduced dissociation. The solid line is the emission spectrum (ex=385nm) after mixing the CPI-and FITC labeled WT streptavidin tetramers atpH 8.6. The dotted line shows a decrease in the donor emission spectrum(CPI) with a corresponding increase in the emission spectrum of thefluorescence energy acceptor (FITC). The emission spectrum of similarlymixed CPI-and FITC-labeled WT streptavidin which had not undergonedenaturation/renaturation, is the sum of the individual spectrum of thetwo components. The tryptophan emission intensity and spectral center ofmass were recovered after guanidine unfolding. The fluorescence spectraare normalized to the integrated intensity of their tryptophan emission,to correct for dilution effects arising from dialysis of the mixedtetramer.

FIGS. 2A and 2B shows ELISA concentration-dependent binding of WTstreptavidin, W79A, and W79F to (FIG. 2A) biotin/BSA at pH 10.0, (FIG.2B) 2-iminobiotin/BSA at pH 10.0. Protein concentrations (tetramer) inμg/ml are plotted on the abscissa, while the alkaline phosphataseactivity indicated by the absorbance at 405 nm are plotted on theordinate.

FIG. 3A shows average EC₅₀ values for the binding of WT streptavidin andTrp mutants to iminobiotin, at pH 8.0 and 10.0. Each mutant and itsassay pH is identified on the abscissa, and the average EC₅₀ in μg ml⁻¹is plotted on a logarithmic y-scale. The asterisk(*) on WxA indicatesthat the reported EC₅₀ is a lower bound, estimated from the(essentially) flat binding isotherm of the Trp->Ala mutants; FIG. 3Bshows ELISA binding isotherms calculated from the 4-parameter fittingfunction for characteristic values of absolute EC₅₀ 's. Estimated K_(a)'s of iminobiotin-protein corresponding to each EC₅₀ are also shown.

FIG. 4A and FIG. 4B show the streptavidin-biotin dissociation rate at298 K, where FIG. 4A is WT streptavidin (filled circle), W79F (opentriangle), and W108F (open square); and FIG. 4B is W120F (open circle).

FIG. 5A shows the binding of W120A and FIG. 5B shows the binding of WTstreptavidin to a biotin column and subsequent elution with 2 mM biotin.

FIG. 6A and FIG. 6B show the purification of chimeric streptavidintetramers: FIG. 6A is 2-iminobiotin affinity chromatography; protein wasbound to iminobiotin column equilibrated in pH 11 binding buffer, washedwith binding buffer, and eluted with pH 4 buffer; FIG. 6B is biotinaffinity chromatography: bound protein from FIG. 6A was incubated withbiotin, and exhaustively ultrafiltered to specifically block with WTsubunits with biotin and passed over biotin column; protein was bound tothe biotin column in PBS and eluted with 2 mM biotin in PBS.

FIG. 7A is a schematic illustration of dissociation across thedimer/dimer interface with subsequent reassembly of heterodimerictetramers. The shading denotes a tetramer with non-identicalcompositions, e.g. mutant binding sites, different fluorescent labels,conjugated drug, etc. FIG. 7B shows a schematic illustration ofdissociation across both the dimer/dimer and monomer/monomer interfacesso that both dimer and monomer intermediate species are present.Subsequent reassembly results in a collection of chimeric tetramerspecies.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides streptavidin and avidin molecularadaptors which are chimeric in that the streptavidin tetramers haveheterologous, but defined, subunit composition and function. In otheraspects of the invention the position of the heterologous subunitswithin the streptavidin tetramer can be controlled. Chimericstreptavidin tetramers are provided by employing genetic engineeringtechniques and biophysical tetramer dissociation/reassembly procedures.The chimeric streptavidin and avidin molecular adaptors provide a widevariety of uses, including new approaches to therapeutic-related drugdelivery, the in vivo delivery of imaging agents, cell sorting andseparations, and diagnostic applications.

The chimeric streptavidin tetramers are produced by a controlled mixingof subunits, where one or more of the subunits have properties that aredesired for a particular utility. For example, a subunit with adecreased affinity for biotin when compared to wild-type streptavidinwill be able to release an anti-neoplastic agent to which it isconjugated more easily than if conjugated to the wild-type streptavidinsubunit. Of course, the individual subunits or dimers thereof can haveproperties other than, or in addition to, an altered binding affinityfor biotin.

The production of the streptavidin subunit having the alteredcharacteristics can be accomplished by several routes depending on theintended use and the characteristic of the subunit which is modified.For example, in some instances subunits, dissociated or nondissociated,may be labeled, directly or indirectly, with a fluorescent orradionuclide label or the like, or linked to another compound, etc., andthen dissociated (if not already dissociated) and mixed with othersubunits as described herein to form the chimeric tetramers.

In certain embodiments described herein the streptavidin subunits have areduced binding affinity for biotin (or iminobiotin), e.g., by means ofamino acid substitutions or deletions in residues of the biotin bindingdomain, and especially by changing the Trp residues at positions 79, 92,108 and/or 120. Such characteristic resulting in the reduced bindingaffinity are accomplished by recombinant DNA techniques, where thespecific amino acid residues of the streptavidin polypeptide arealtered, e.g., by site-directed mutagenesis. Although examples ofmutations which result in diminished affinity of the streptavidinpolypeptide for biotin are described in the Example section below, wherealterations at Trp79, Trp92, Trp108 and Trp120 result in a diminishedbinding, additional alterations based on the teachings hereof may beemployed. For example, any of the amino acids other than Trp may besubstituted at the selected position(s). The reduced binding affinitiesof the altered streptavidin for biotin (and/or iminobiotin) willtypically be less than approximately 1×10¹³ M⁻¹, sometimes less thanabout 1×10¹² M⁻¹, sometimes less than or equal to about 1×10¹¹ M⁻¹, andin some cases as low as about 1×10¹⁰ M⁻¹ or lower, e.g., 1×10⁷ M⁻¹.

In addition to engineering changes in streptavidin's biotin binding sitefor biotin to reduce the binding affinity, on-rate or off-rate, othermutations to subunits or dimers of streptavidin can be made. Theseinclude, for example, engineering of enhanced dissociation equilibria todestabilize the dimer/dimer interface through site-directed mutagenesisof side-chains at the dimer/dimer interface. For example, thestreptavidin monomer may contain at least one mutation in the amino acidresidues situated at the dimer/dimer interface. In this case a disulfidebond can be engineered to connect specific subunits and define thestoichiometry of the dissociated species. Alternatively, an enhancedsensitivity to hydrostatic pressure can be engineered, without alteringsolution stability, at the dimer/dimer interface. Other mutations can bemade to the subunits to permit attachment of drugs, linkers, enzymes,labels, etc.

Once the streptavidin subunits have been modified to possess the desiredcharacteristic, different subunits, e.g., modified and wildtype, aremixed to reform a heterologous or chimeric streptavidin tetramer havingone, two or three modified subunits. Homologous tetramers havingmodified subunits in each position are formed by refolding the modifiedsubunits and do not require mixing. The subunits can be cross-linked toform diners and the same or different dimers reassociated to form theheterologous tetramer, or the subunits can be simply mixed at themonomer level to form 1:3, 2:2 and 3:1 modified tetramers. Thus, a widevariety of combinations are made possible by the present invention.

Subunit mixing within the streptavidin tetramer can be monitored by avariety of different techniques. In one technique, a fluorescence assayis used to follow the mixing of oligomeric protein subunits afterdissociation by high pressure, as generally described in Erijman andWeber, Biochemistry 30: 1595-1599 (1991), incorporated by referenceherein. In this assay, one population of the oligomeric protein islabeled with a fluorescence energy transfer donor, e.g.,7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, and asecond population is labeled with a complementary energy transferacceptor, e.g., fluorescein isothiocyanate. Sulfhydryl-selectivederivatives of these fluorophores can be used to label a site-directedmutant which has been constructed to provide a stoichiometric labelingsite for thiol-specific small molecules, e.g., the Asn49Cys mutant,which will provide one fluorophore donor or acceptor per subunit. Whenthe two populations are mixed at ambient conditions, the fluorescenceemission spectrum is simply the addition of the individual donor andacceptor spectra. This is because the dissociation rates of oligomericproteins are generally negligible and so there is no mixing of the twopopulations. When the mixed proteins are exposed to denaturants wherethe dissociated states are populated, and then brought back to ambientconditions, the fluorescence emission is altered by energy transfer. Theexcitation wavelength for energy transfer studies with thisdonor/acceptor pair is 385 nm and the emission spectra are collectedfrom 400 nm to 600 nm. This energy transfer spectrum is diagnostic ofmixing between the donor-labeled subunits with acceptor-labeledsubunits, where some of the reassociated oligomers have the donor andacceptor labels present on the same molecule.

Thus, the monitoring technique can be used to demonstrate subunit mixingafter denaturant-induced tetramer dissociation. In one example, arecombinant streptavidin population was labeled with the coumarinfluorophore, and a second population with a fluorescein derivative.These populations were mixed and the fluorescence emission spectrumshown by the solid line in FIG. 1 was obtained. The sample was thenbrought to 6 M guanidinium thiocyanate for 1 hr and then dialyzed backto the starting conditions. The refolding was nearly quantitative asevidenced by the return of the tryptophan fluorescence intensity andemission maximum to those measured under the starting conditions. Thedye emission spectra were altered in a manner consistent with theemergence of energy transfer between the coumarin and fluorescein dyes(dotted line, FIG. 1). This experiment provides evidence for subunitmixing after dissociation of the tetramer.

Monitoring the chimeric tetramers of the protein population can also beperformed by electrospray mass spectrometry (ESMS). Recent developmentsin ESMS have allowed the noncovalent association in multisubunitproteins to be preserved, allowing for the detection of the entire,multisubunit complex. See Light-Wahl et al., J. Amer. Chem. Soc.115:5869 (1993); Light Wahl et al., J. Amer. Chem. Soc. 116:5271 (1994);and Schwartz et al., J. Amer. Soc. Mass Spectrom. 5:201-204 (1994) eachof which is incorporated herein by reference. The chimeric streptavidintetramers are analyzed by ESMS under conditions that favor preservationof noncovalent association. Corroboration of tetramer composition can beobtained by analyzing the protein under conditions that favordissociation of the tetramer into monomers.

Selective pressure dissociation can also be used for the streptavidintetramer, particularly where the dimer/dimer interface has beengenetically engineered to be susceptible to such dissociation. Highpressure techniques are used to dissociate the tetramer into subunitsunder equilibrium conditions that permit efficient mixing ofheterologous subunit populations. Pressure dissociation is generallyvery gentle and reversible because the physical driving force forpressure induced dissociation is the difference in volume between thetetramer and the isolated subunits. The application of pressure favorsthe side of the equilibrium with lowest net volume, and in most casesthe individual subunits (monomers or dimers) will have a lower netvolume than the oligomeric state (tetramer). Thus, application ofhydrostatic pressure will generally shift the association equilibriumtoward the dissociated states. Thus, dissociation and subunit mixing canbe controlled without significantly altering the tetramer stabilityunder ambient conditions. In addition, by introducing charge pairs atspecific interfaces via site-directed mutagenesis, pressure-induceddissociation to either the monomer or to the dimer state can beeffectuated. This is shown with the dimer-dimer interface, which isminimal and structurally well defined, and can be applied to engineerthe pressure sensitivity of the monomer-monomer interface. Byengineering the two interfaces (dimer-dimer and monomer-monomer) to havedistinct pressure sensitivities, this approach provides means forcontrolling the species (monomer or dimer) available for mixing--andthus control the architecture of the chimeric binding sites within mixedtetramers. The pressures necessary to mix tetramer subunits aredetermined using the fluorescence energy transfer and mass spectrometryapproaches. Pressure ranges are established that dissociate the tetramerinto dimers but not monomers, and that dissociate the tetramer intomonomers.

In another aspect, tetramer subunit connectivity is altered bysite-directed mutagenesis to introduce residues capable of formingdisulfide bonds, and thus control tetramer reassembly via the disulfidebonds. For example, spatially proximal β-carbon positions of thestreptavidin protein-protein interfaces are determined where thedistance and dihedral geometry is appropriate for engineering disulfidebonds, e.g., positions across the monomer/monomer interface anddimer/dimer interface. A covalent bond is preferably engineered betweenthe monomers in the dimer, for example, at position H127. It is thendemonstrated that when dissociation to dimers occurs, a labeled tetramerarising from the tagged disulfide bonded dimer returns.

The mixed streptavidin tetramers with defined subunit composition whichare constructed according to the present invention find use in a widevariety of drug delivery and imaging, cell sorting and separationtechnologies. Streptavidin is used in a number of crucialseparations/cell-sorting technologies. The ability to reconstruct mixedstreptavidin tetramers with defined subunit orientation as provided bythe present invention greatly improves these molecular tools. Forexample, two subunits can be labeled with fluorescent dye markerswithout disturbing the affinity of the remaining two sites, therebyensuring that the label is on one side of the tetramer away from theother two binding sites, and thus not sterically interfering withbinding affinity. The site-directed mutants of the invention providelabeling sites for markers or probes, e.g., luminescent agents,radiolabels, enzymes, chromophores, ferritin, hemocyanin, macromolecularcarriers, etc., and these can similarly be mixed with wild-type subunitsto form chimeric tetramers. The site-directed mutants can also be usedto provide sites for immobilizing streptavidin to surfaces such asmagnetic beads or chromatography supports and these can be mixed withwild-type subunits to yield sterically optimized cell separationscomponents. Further, the site-directed mutants designed for orderingstreptavidin at surfaces can be combined with site-directed mutants thatlower the binding affinity, thereby providing components that aresterically optimized and which have affinity that permits more gentleelution of bound cells. Thus, the applications of the modified moleculesof the instant invention include, for example, affinity chromatography,affinity cytochemistry, histochemistry, diagnostics, signalamplification, blotting technology, bioaffinity sensors, gene probes,drug delivery, crosslinking agents, affinity targeting, affinityperturbation, fusogenic agent, immobilizing agent, selective retrieval,and selective elimination, among others.

For example, methods for immunoselection of cells using avidin andbiotin, in which the lower affinity mutants of the present invention areparticularly useful, are described in detail in Berenson et al., U.S.Pat. No. 5,215,927, incorporated herein by reference. In one method forseparating a target substance such as a hematopoietic stem cell from aheterogeneous suspension containing the target substance, a suspensionsuch as a cell culture, bone marrow, peripheral blood or cord blood isreacted with a biotinylated binding component which binds to the targetsubstance, thereby forming a biotinylated targeted substance complex.The biotinylated binding component can be an antibody, polyclonal,monoclonal or binding fragment thereof, that binds specifically to CD34+hematopoietic stem cells. The suspension containing the biotinylatedtarget substance complex is exposed to a modified streptavidin moleculeof the present invention, e.g., a chimeric tetramer comprising at leastone modified monomer but less than four modified monomers. Forconvenience, the exposing step can be performed in a column in which thestreptavidin tetramer of the invention has been adsorbed to a solidphase. The biotinylated target substance complex is separated from thesuspension by means of the lower affinity (or increase in dissociationrate constant) of the modified streptavidin monomer units to recover thetarget substance in enriched form.

For drug delivery, imaging, and other such uses, a chimeric streptavidintetramer comprising, e.g., two higher affinity and two lower affinitymodified monomers, is loaded with a biotinylated molecule (e.g.,biotinylated antibody) such that the biotinylated molecule isselectively partitioned to the higher affinity subunits by virtue of thefast off-rate of the lower affinity subunits. The biotinylatedtarget/chimera complex is targeted to the desired site, either in vivoor ex vivo, via the antibody. A biotinylated imaging agent or drug isadministered and captured by the lower affinity subunits, whereby thetargeted cells or tissues are thus exposed to the drug or label for thedesired site-specific therapeutic or diagnostic activity. In anotheraspect, a biotinylated targeting agent is administered, followed by astreptavidin chimera of the present invention having high and loweraffinity binding sites. The high affinity sites bind to the biotin atthe target site. A biotinylated imaging or therapeutic agent is thenadministered, which binds at the available lower affinity streptavidinsites. This results in a real time release from the streptavidinmolecule of the therapeutic or imaging agent at the targeted site. Inyet another embodiment, the chimeric streptavidin tetramer of, e.g., twohigher affinity and two lower affinity modified monomers, is loaded witha biotinylated molecule such as an antibody (or receptor, ligand etc.)at the higher affinity subunits, and the lower affinity sites are loadedwith biotinylated drug, therapeutic protein or imaging label. Thechimeric complex is targeted to the desired site or cells in vivo or exvivo via the targeting component, where the lower affinity component isreleased to impart the desired therapeutic or diagnostic activity. Inyet another example, the lower affinity subunits conjugated to enzymes(e.g., alkaline phosphatase) that are suitable for conversion ofprodrugs (e.g., etoposide phosphate) can be mixed with higher affinitysubunits conjugated to antibodies or other targeting components, suchthat when administered to the patient or cell collection the antibodydelivers the chimeric complex to the desired site(s) and the enzymeactivates prodrug which is administered to the patient or cells.Suitable prodrugs, enzymes, and methods of administration are describedin Senter et al., U.S. Pat. No. 4,975,278, incorporated herein byreference.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE 1

This Example describes the construction of streptavidin having aminoacid modifications of the biotin binding site, which modifications areshown to result in a reduced binding affinity for iminobiotin andbiotin.

Design and Construction of a Synthetic Gene for Core Streptavidin

The program GCG (Genetics Systems Group Inc., Madison, Wis., 1991) wasused to generate a nucleotide sequence from the amino acid sequence ofcore streptavidin, defined as residues 13-139 of the naturally occurringprotein (for the sequence of streptavidin see Argarana et al., Nucl.Acids Res. 14: 1871-1882 (1986), and U.S. Pat. No. 4,839,293, each ofwhich is incorporated herein by reference). The synthetic gene for corestreptavidin incorporates favorable E. coli codon usage (deBoer et al.,Maximizing Gene Expression, eds., Reznikoff et al. (Butterworth,Stoneham, Mass., USA) pp. 225-285 (1986)), a consensus ribosome bindingsite (Shine et al., Nature 254: 34-38 (1975)), an initiating methioninecodon, translational stop codons, and a number of unique restrictionendonuclease recognition sites spaced evenly throughout the length ofthe gene. These features were incorporated into the gene design tofacilitate convenient generation of site-directed mutants by cassettemutagenesis as well as expression from plasmids lacking their ownribosome binding site.

Single stranded oligodeoxyribonucleotides were synthesized commercially(Oligos Etc.) and purified by gel filtration. The core streptavidin genewas constructed in three segments, which were flanked by the followingrestriction endonuclease recognition sites: EcoRI/XbaI, XbaI/HincII, andHincII/HindIII, respectively. For each segment, individualoligodeoxyribonucleotides were 5'-phosphorylated, annealed, and ligatedinto a separate PUC18 plasmid that had been linearized with theappropriate pair of restriction endonucleases. (Note that except whereexplicitly stated otherwise, protocols for standard procedures such asplasmid isolations, ligations, transformations, and digestion withrestriction endonucleases are according to Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 2nd Ed.(1989)) or from instructions provided by commercial suppliers ofreagents and kits.) The plasmids were subsequently transformed intoDH5a, or Novablue (Novagen Inc., Madison, Wis.) E. coli cells. Followingselection of clones containing the appropriately sized inserts by colonyPCR, plasmid constructs were sequenced using dye terminator chemistrywith fluorescent detection of sequencing products (Applied Biosystems,Foster City, Calif.). The three DNA segments comprising the corestreptavidin gene were isolated and ligated into a single pUC18 plasmid,which was transformed into Novablue cells. At this point, the entiregene was sequenced to confirm the nucleotide sequence. The corestreptavidin gene was then subcloned into the E. coli expression vector,pET-21a (Novagen Inc.), as an NdeI/HindIII segment and maintained inNovablue cells.

Site-Directed Mutagenesis of Core Streptavidin

W79A, W79F, W108A, and W108F site-directed mutants of WT streptavidinwere created by PCR mutagenesis, using mutagenic primers incorporatingthe desired codon change (Sligar et al., Meth. Enzymol., 206: 31-49(1990)). The PCR mutagenesis primers were as follows:

W79A (SEQ ID NO:1): 5' TTT CGC AGC AAC GGT CCA ACC CAG AGC GGT TCC AGA A3';

W79F (SEQ ID NO:2): 5' TTT GAA AGC AAC GGT CCA ACC CAG AGC GGT TCC AGA3';

W92A (SEQ ID NO:3): 5' ACC GCG TCT GGT CAG TAC GTT GGT GGT GCT GAA 3';

W92F (SEQ ID NO:4): 5' CC TTC TCT GGT CAG TAC GTT GGT GGT GCT G 3';

W108A/F (SEQ ID NO:5): 5' GGT CGT ACC GGA GGT CAG CAG CGA CTG G 3' and(SEQ ID NO:6): 5' GGT CGT ACC GGA GGT CAG CAG GAC CTG G 3';

W108F (SEQ ID NO:7): 5' AG TTC TTG TTG ACC TCC GGC ACC ACC GAA GCT AACGCT TGG 3'.

W120A and W12OF were created by cassette mutagenesis, using thefollowing sequences: Sense Strand 5' -> 3' (SEQ ID NO:8 and SEQ IDNO:9): CGC GGC TAA ATC CAC CCT GGT TGG TCA CGA CAC CTT CAC CAA AGT TTTAA; and Antisense strand 5' -> 3' (SEQ ID NO:10 and SEQ ID NO:11): AGCTT TTA TTA GGA AGC TGC AGA CGG TTT AAC TTT GGT GAA GGT GTC GTG ACC AACCAg GGT GGA TTT AGC AA

Oligodeoxyribonucleotides, chemically 5'-phosphorylated during synthesis(IDT Inc.), spanning the MluI/HindIII endonuclease restriction siteswith degenerate codons at residue 120 incorporating the Trp->Ala/Phemutation, were annealed, ligated into MluI/HindIII linearized pUC18containing the core streptavidin gene, and transformed into competentNovablue cells. Clones containing mutant plasmid were identified by theinsertion of a PstI restriction site upon successful ligation of themutagenic insert into the core streptavidin gene. DNA sequencing ofplasmids containing a PstI restriction site was used to identify clonescontaining either the Phe or Ala mutation. The mutant streptavidin genes(W120A or W120F) were subcloned into pET21a as an NdeI/HindIII segment,and maintained in Novablue cells.

Expression of Streptavidin and Mutants in E. coli

The expression plasmid pET-21a containing the streptavidin gene,transformed into the expression host, BL21(DE3) (Novagen, Inc.), wascultured overnight at 37° C. with shaking, in 10 ml LB mediumsupplemented with 100 μg/ml ampicillin. The culture was then centrifugedat 4500×g for 5 min, the cell pellet resuspended in fresh 10 ml LBmedium and used to inoculate 6.5 L of 2×YT medium supplemented with 100μg/ml ampicillin in shaker flasks. The culture was incubated withshaking at 37° C. until the absorbance at 600 nm reached 1.0, at whichpoint isopropyl β-D-thiogalactopyranoside was added (1 mM) to induceprotein expression. Cells were cultured for a further 3 hrs, after whichthey were harvested by centrifugation at 4500×g for 10 min. The cellpellets were stored at -70° C. until further use. Quantitation ofprotein bands in SDS-polyacrylamide gel electrophoresis (PAGE) ofinduced BL21(DE3) cell lysates by laser densitometry revealed thatstreptavidin constituted 15-20% of the total cellular protein.

Isolation and Purification of Expressed Streptavidin and Mutants

The frozen cell pellet was thawed, resuspended in 200 ml 50 mM Tris HCl,pH 8.0/0.75 M sucrose/l mM phenylmethylsulfonyl fluoride (PMSF), andruptured by sonication. The lysed cells were incubated at roomtemperature for 15 min with DNAseI (10 μg/ml)/RNAseA (10 μg/ml)/MgCl₂(10 mM), and centrifuged at 22000×g for 30 min. The insoluble fractionwas repeatedly washed with 50 mM Tris, pH 8.0/10 mM EDTA/1.5 M NaCl/l mMPMSF/0.5% (v/v) Triton-X-100 to solubilize membrane proteins. The finalwhite pellet, largely comprising streptavidin inclusion bodies, wasapproximately 70% pure, as determined by SDS-PAGE. The inclusion bodieswere dissolved in 6M guanidine HCl (500 ml), pH 1.5, to a concentration≦50 μM (streptavidin monomer) and dialyzed against 20 liters 50 mM TrisHCl, pH 8.0/150 mM NaCl/10 mM EDTA/0.1 mM PMSF/0.5 mM benzamidine HClover 24 h with one 20 L change of dialysis buffer. The dialysate wascentrifuged, vacuum filtered through 0.45 μM filters, and concentratedin a stirred ultrafiltration cell (Amicon Inc., Danvers, Mass.). Finalconcentration to a few ml utilized Centriprep-30 centrifugalconcentrators (Amicon Inc.). Insoluble, aggregated protein left overafter dialysis can be refolded several times by following the aboveprotocol.

WT Streptavidin and the Trp->Phe mutants were purified by affinitychromatography using iminobiotin-agarose (Pierce, Naperville, Ill.)(46,47). The yield of affinity-purified WT streptavidin was .sup.˜ 10-20mg per liter of 2×YT culture. The lowered affinity of the Trp->Alamutants for iminobiotin precluded its use for purification. Instead, thesamples were applied to a DEAE-SepharoseFF column (1.5×5 cm)equilibrated with 20 mM Tris HCl, pH 7.0 (Pharmacia, Piscataway, N.J.).Under these conditions, streptavidin does not bind to the column and iseluted in the void volume, though reasonable purification is affordeddue to the binding of DNA and most of the contaminant proteins from E.coli. The streptavidin-containing fractions were pooled, concentratedand equilibrated in 20 mM Tris, pH 8.5 and passed over aDEAE-SepharoseFF column (1.5×5 cm) equilibrated in the same buffer.Streptavidin bound under these conditions and was then eluted byapplying a linear 0-0.3 M NaCl gradient. Streptavidin-containingfractions were pooled, concentrated and stored at 4° C. for further use.These two chromatography steps afforded homogeneous protein, as verifiedby SDS-PAGE.

Refolded, affinity-purified WT streptavidin was characterized byamino-acid compositional analysis and N-terminal sequencing. Theexperimentally-determined composition and the N-terminal sequence,respectively, of the recombinant protein agreed with the calculatedcomposition and sequence of core streptavidin. SDS-PAGE of heatdenatured protein showed that the monomers for each Trp mutant were thesame size as WT streptavidin monomers, and that the purification methodsemployed for WT streptavidin and the Trp mutants yielded homogeneousprotein, with contaminant proteins below the detection limits ofCoomassie staining. ESMS of WT streptavidin and Trp mutants revealedthat the experimentally-determined mass of WT streptavidin and Trpmutants were correct. The similar rates of migration of WT streptavidinand Trp mutants in native-PAGE indicated that the refolded mutantsself-assembled to form tetramers in solution, similar to WTstreptavidin. The binding of fluorescein-biotin to WT streptavidin andthe Trp mutants ranged from 0.85 to 1.1 (on a subunit basis) uponsaturation, close to the stoichiometric ratio of 1.0 predicted forbinding of one biotin per streptavidin subunit.

Characterization of Streptavidin and Mutants

SDS-PAGE analysis was carried out using precast Miniprotean 10-20%gradient gels (BioRad Inc., Richmond, Calif.) with a discontinuousbuffer system (Laemmli, Nature 227: 680-685 (1971)). Samples were boiledfor 15 min in the presence of SDS before electrophoresis to dissociatestreptavidin tetramers. Native-PAGE was performed using the above systemby omitting SDS in the sample application buffer and the gel runningbuffer, as well as the heat denaturation of proteins beforeelectrophoresis. The gels were stained with 0.25% (w/v) Coomassie R-250,dissolved in 45% methanol (v/v), 10% acetic acid (v/v). Theconcentration of WT streptavidin was determined by absorbance at 280 nmusing an extinction coefficient (e₂₈₀) of 34000 M⁻¹ cm⁻¹ for the subunit(Sano et al., Proc. Natl. Acad. Sci. USA 87: 142-146 (1990)).

Concentrations of the mutants were determined by the method of Gill etal., Anal. Biochem. 182: 319-326 (1989), using e₂₈₀ of WT streptavidinas reference. Protein electrospray mass spectrometry (ESMS) was carriedout on an API III electrospray mass spectrometer (PE/Sciex, Thornhill,Ontario). The biotin-binding stoichiometry of WT streptavidin andmutants was determined in solution by the quantitative quenching in thefluorescence of 5-((N-(5-(N-(6-(biotinyl)amino)hexanoyl) amino)pentyl)thioureidyl)fluorescein (Fluorescein-biotin, Molecular Probes, Eugene,OR) upon titration with protein.

ELISA Assays

A modified version of the enzyme assay reported by Bayer et al., Anal.Biochem. 154: 367-370 (1986), was used to examine theconcentration-dependent binding of WT streptavidin and mutants withbiotin and 2-iminobiotin. 2-Iminobiotin conjugated to bovine serumalbumin (iminobiotin/BSA) was synthesized by reacting a twenty-foldmolar excess of 2-iminobiotin N-hydroxysuccinimide ester (Sigma) with100 mg BSA in 100 mM NaHCO₃, pH 8.3 for 12 hr at 4° C. with end-over-endstirring, followed by dialysis and gel filtration (G-25, Pharmacia) toseparate iminobiotin/BSA from unreacted 2-iminobiotin. Biotin/BSA(Pierce) or iminobiotin/BSA at a concentration of 10 mg/ml were adsorbedovernight at 4° C. in 15 mM Na₂ CO₃, pH 9.6, in microwell plates (100 μlper well). The next day the microwell plates were incubated with 200 μlper well of blocking buffer at pH 8.0 or pH 10.0 (50 mM sodiumphosphate, pH 8.0 or 50 mM Na₂ CO₃, pH 10.0/100 mM NaCl/0.5% (w/v)BSA/0.05% (v/v) Tween-20) for at least 2 hr at room temperature and thenincubated in ten-fold serial dilutions of 100 μg/ml WT streptavidin ormutants (100 μl/well) for 2 hr at room temperature, rinsed with blockingbuffer at pH 8.0 or pH 10.0 (200 μl/well), and incubated for 1 hr atroom temperature in 2×10⁴ dilution of primary antistreptavidinantibodies (Sigma) in pH 8.0 or pH 10.0 blocking buffer (100 μl/well).The plates were rinsed three times with 200 μl per well blocking bufferat the assay pH, incubated with secondary anti-IgG/alkaline phosphataseconjugate (Sigma) for 1 hr at room temperature, rinsed three times withblocking buffer, and assayed for alkaline phosphatase activity at pH10.0. Every plate assayed had triplicates for each proteinconcentration. The data were processed on Mathcad (Mathworks) todetermine the equivalent bulk concentration at 50% saturation of binding(EC₅₀) values using a published 4-parameter nonlinear fitting algorithmshown below (Jin et al., J. Mol. Biol. 226: 851-865 (1992)):

    y=a+(d-a)/[1+exp(b(c-x))]

where a, b, c, and d are the adjustable fitting parameters;x=streptavidin concentration (μg/ml); y=absorbance at 405 nm. AbsoluteEC₅₀ in μg ml⁻¹ are given by the value of the parameter c for the bestfit of the fitting function to the binding isotherm. Relative EC₅₀values reported in Table I are EC₅₀ (mutant)/EC₅₀ (WT streptavidin).

Equilibrium Binding of Biotin

³ H-biotin at a concentration of 2 nM (WT, W79A, W120A) or 0.2 nM (WT,W120F) was incubated in aliquots of serially diluted protein for 2 h.The free ligand was then separated from the protein-bound ligand byeither Microcon-30 or Centriprep-30 centrigual ultrafiltration devices(Amicon, Inc.). Typically, 0.1-1 ml of the free ligand solution or theprotein-ligand mixture was added to 18 ml liquid scintillation cocktail(Ecolume, ICN Biomedicals, Inc., Costa Mesa, Calif.) and assayed on aliquid scintillation counter (Beckman Instruments, Inc., Fullerton,Calif.). The K_(a) was determined from a nonlinear curve fit of thefraction of protein-bound ligand versus the free protein concentration.

With biotin as the ligand, the concentration-dependent binding isothermsof WT streptavidin and all Trp mutants are largely identical in theELISA assay at both pH 8.0 and 10 (FIG. 2A shows the binding isotherm ofWT streptavidin and the W79A/F mutants). However, with iminobiotin/BSAas the ligand (FIG. 2B for WT streptavidin and the W79A/F mutants) thebinding isotherms indicate marked differences in the affinities of WTstreptavidin and the mutants, with the EC_(50's) in the orderWT<Phe<Ala. The absolute and relative EC₅₀ for the binding of the WTstreptavidin and the Trp mutants to iminobiotin, at pH 8.0 and 10.0 aresummarized in Table I and FIG. 2.

                  TABLE I                                                         ______________________________________                                        EC.sub.50 results for binding of WT streptavidin and Trp                        mutants by 2-iminobiotin at pH 8.0 and pH 10.0                                                     Absolute EC.sub.50                                                                     Relative EC.sub.50                              Protein pH (μg ml.sup.-1) (Mutant/WT)                                    ______________________________________                                        WT*     8/10        0.03 ± 0.02                                                                          1.0                                               WT  8 0.046 ± 0.03 1.0                                                     WT 10 0.052 ± 0.013 1.0                                                    WxA** 8/10 ≧100 ≧2000                                           W79F  8  8.27 ± 3.01 180                                                   W79F 10  0.14 ± 0.01 2.7                                                   W108F  8  9.6 ± 0.74 209                                                   W108F 10  0.12 ± 0.07 2.3                                                  W120F  8 10.27 ± 0.5 223                                                   W120F 10  4.03 ± 1.91 78                                                 ______________________________________                                         The absolute EC.sub.50 values (μg/ml.sup.-1) were derived from the         nonlinear least squares fit of at least three independent measurements of     the binding isotherm. The relative EC.sub.50 values are normalized to the     average (absolute) EC.sub.50 of WT streptavidin at pH 8 or pH 10,             respectively.                                                                 *Biotin is the ligand. Six independent isotherms of WT binding to             biotin/BSA were fitted to determine the EC.sub.50.                            **The complete binding isotherm off the Trp >Ala mutants could not be         determined, leading to a lower bound of their EC.sub.50.                 

These experimental results indicated the Ala mutants were accessible forequilibrium biotin binding assays. A spin-column assay with a 30,000 MWcutoff filtration membrane was used to quantitate the partitioning of ³H-biotin between the free and bound states. This assay provides a usefulestimate of the K_(a) when the affinities lie between 10⁶ -10⁹. Theconcentration-dependent binding isotherm for W12OF was at thetight-binding limit (K_(a) >10⁹) at the lowest experimentally accessibletotal biotin concentration (0.2 nM) and identical to wild-typestreptavidin. While the W108A mutant protein proved to be unstable inthis assay at the necessary concentrations, the W79A and W120A mutantsyielded complete concentration-dependent binding isotherms from whichthe K_(a) was determined to be 4.3×10⁷ M⁻¹ and 8.6×10⁶ M⁻¹,respectively. These experimentally determined K_(a) estimates provideindependent confirmation of the ΔK_(a) 's estimated from the ELISAassays as further discussed below.

Order of magnitude determinations of ΔK_(a) for the mutants wereestimated from the EC₅₀, given the known K_(a) 's of some of theseligand-protein partners. The subsequent analysis is based on theexperimentally-determined K_(a) of 2.5×10¹³ M⁻¹ of biotin-WTstreptavidin and the K_(a) of =10⁸ M⁻¹ for iminobiotin/WT streptavidinat pH 10.0, estimated from the experimentally-determined value foriminobiotin/avidin. These numbers are further supported by theindependently determined biotin-binding affinities of the W79A and W120Amutants, which agree well with the ELISA estimates.

The ELISA assay is insensitive to the ΔK_(a) in ligand-protein bindingwhen the K_(a) are higher than -10⁷ M -1, i.e., the ELISA bindingisotherms for ligand-protein partners are indistinguishable when theK_(a) 's are in the range 10⁷ -10¹³ M⁻¹. This assumption is supported bythe following observations: the experimentally measured EC₅₀ for WTstreptavidin/biotin is identical to the EC₅₀ of WTstreptavidin/iminobiotin at pH 10.0, despite the marked differences inthe K_(a) of WT streptavidin/biotin (2.5×10¹³ M⁻¹), and that ofiminobiotin/WT streptavidin at pH 10.0 (˜10⁸ M⁻¹). The similar EC₅₀ 'sfor these two ligands suggest that ELISA is generally insensitive toΔK_(a) in the range of 10⁸ -10¹³ M⁻¹. Furthermore, the binding of WTstreptavidin by iminobiotin is pH-sensitive, and the K_(a) of WTstreptavidin/iminobiotin is likely to be an order of magnitude lower atpH 8.0 than at pH 10.0, as is the experimentally-determined ΔK_(a) forthe closely related iminobiotin-avidin system. Thus, an upper limit of10⁷ M⁻¹ for the sensitivity of ELISA towards ligand K_(a) can beestablished. The similar biotin EC₅₀ values measured for WT streptavidinand all of the Trp mutants similarly suggests that the K_(a) of the Trpmutants for biotin are in the 10⁷⁻¹⁰ ¹³ M⁻¹ range.

However, when the ligand affinity is lowered into the accessible rangeof this ELISA assay by using iminobiotin as the ligand, marked changesin the EC₅₀ 's are observed for the Trp mutants. The relative EC₅₀results for the Trp mutants are summarized in FIG. 3, and illustratetheir increased iminobiotin EC₅₀ 's. W79F and W108F display ˜2˜3-foldgreater EC₅₀ 's at pH 10.0 compared to WT streptavidin; when the assaypH is lowered to 8.0, their EC₅₀ 's dramatically increased by two ordersof magnitude, consistent with the pH-dependent decrease in the affinityof iminobiotin for streptavidin. These results suggest that the affinityof W79F and W108F mutants for iminobiotin is less than one order ofmagnitude lower than WT streptavidin at pH 10.0 (K_(a)˜107-108 M-1).Lowering the assay pH to 8.0 further decreases the K_(a) to ˜10⁶ M⁻¹),compared to W79F and W108F, as shown by its 100-fold greater relativeEC₅₀ at pH 10.0, and a weaker pH-dependence, shown by the smallerincrease in relative EC₅₀ at pH 8.0 (relative EC₅₀ ˜200 at pH 8).

Mutating Trp>Ala results in larger changes in 2-iminobiotin bindingaffinities, so that only a lower bound of their EC₅₀ s can be estimated(relative EC₅₀ ≧2000). The EC₅₀ 's for the Trp->Ala mutants bound toiminobiotin may be somewhat overestimated by a disproportionate loss ofthe bound protein during multiple washing steps in ELISA. Since the Alamutants display a biotin-binding isotherm that is indistinguishable fromthat of WT streptavidin, which sets a maximum ΔK_(a), the ΔK_(a) 's ofthe Ala mutants are estimated to fall between 10⁴ -10⁶. This result issupported by the direct estimation of K_(a) for the W79A and W120Amutants, which places these affinities at 10⁷ M⁻¹.

The present analysis assumes that the EC₅₀ differences for iminobiotinreflect similar differences for biotin, which is supported by thecalculations of Miyamoto and Kollman, that the difference in theabsolute free energies of binding for the two ligands are largely due tothe differences in the solvation energies of the two ligands rather thanlarge differences in ligand-protein interaction free energies (Miyamotoand Kollman, Proteins 16: 226-245 (1993), and Miyamoto and Kollman,Proc. Natl. Acad. Sci. USA 90: 8402-8406 (1993)). The ΔK_(a) estimatesfrom the 2-iminobiotin ELISA binding isotherms are closely corroboratedby the direct estimates of the biotin K_(a) for the W79A, W120A andW120F mutants. Iminobiotin is thus a good reporter for intrinsicstreptavidin-biotin interactions, consistent with the fact that thetryptophans do not directly interact with the structurally alteredureido moiety.

These results, and notably the ranking of affinities in the order of thearomaticity of the side chains Trp (WT)>Phe>Ala and the associatedmagnitude of the changes in the EC₅₀ for binding to iminobiotin,indicate that altering the aromatic content of the contact residuesgreatly -impacts the absolute free energy of binding. Thus, partialretention of side chain aromaticity by altering Trp to Phe results in a10¹ -10² increase in iminobiotin EC₅₀ 's, which is indicative of asimilar decrease in their K_(a) for biotin. Complete abolition of thearomatic side chain by mutating Trp to Ala results in considerablygreater EC₅₀ 's, which is indicative of a ΔK_(a) (biotin) .sup.˜ 10³lower than WT streptavidin. These results suggest that the contributionof a Trp side chain to the absolute free energy of biotin binding couldbe as much as .sup.˜ 4 kcal/mol; since four Trp residues contact biotin,their overall contribution to the absolute free energy of binding biotinis substantial.

The equilbrium binding enthalpies, ΔH°, and the heat capacities, ΔCp°,have also been engineered with the streptavidin mutants (Table II). Heatcapacities were determined as generally described in Murphy et al.,Proteins: Structure, Function and Genetics 15:113-120 (1993),incorporated herein by reference. As can be seen, there are examples ofboth increased and decreased ΔH° and increased and decreased ΔCp°. Theheat capacities relate the temperature dependence of the bindingenthalpy, which contributes significantly to the binding affinity.Alterations in the heat capacity are thus important in applicationswhere temperature is used as a variable to control the biotin affinity.

                  TABLE II                                                        ______________________________________                                        Protein     .increment.H° (kcal/mol)                                                          .increment.Cp° (cal/molK)                       ______________________________________                                        WT          -24.5      -345                                                     W79A -18.5 -357                                                               W79F -25.9 -266                                                               W108F -23.5 -386                                                              W120F -19.4 -303                                                              W120A -12.8 -272                                                            ______________________________________                                    

EXAMPLE II

This Example demonstrates the contribution of binding-site tryptophanresidues to the biotin off rate and activation thermodynamics. Thedissociation rate constants of streptavidin mutants W79F, W108F andW120F indicate these Trp contacts are important in regulating thedissociation rate.

The streptavidin-biotin dissociation rate was determined for WTstreptavidin, W79F, W108F, and W120F as follows. 8,9-³ H biotin (4.8 μl,21 μM, NEN/Dupont) was added to 10 ml PBS, pH 7.4, 1 mM EDTA, containingWT streptavidin or one of W79F, W108F or W120F mutants at aconcentration of 0.5 μM and incubated for 10 min followed by addition ofnon-radioactive biotin (20 mM) to a final concentration of 50 μM.Aliquots (0.5 ml) of this mixture were centrifugally ultrafilteredthrough a 30,000 MW cutoff filter (Microcon-30, Amicon Inc., Beverly,Mass.) to separate the unbound biotin from the protein-ligand complex.Fifty μl of the filtrate were mixed with 10 ml scintillation cocktail intriplicate and assayed for radioactivity in a liquid scintillationcounter (LS-7000, Beckman Instruments, Fullerton, Calif.). The averageradioactivity of the filtrate in cpm at each time point andradioactivity of the protein-ligand complex before addition of coldbiotin (a) allowed the first order rate constant of the dissociation ofprotein-ligand complex (k_(off)) to be determined from the plot ofln(a-x/a) [ln(fraction bound)]. Standard deviations were determinedbased on three independent measurements. Control experiments where nocold biotin was added yielded <2% of the total radioactivity in theultrafiltrate, demonstrating that all ³ H biotin was initially bound.

The results, as shown in FIG. 4, indicate that biotin-bound wild-type(WT) core streptavidin and the site-directed mutants W79F, W108F, andW120F display monoexponential, first order, dissociation kinetics. Thefirst order dissociation rate constants (k_(off)) are summarized inTable III. The k_(off) of recombinant core WT streptavidin (k_(off)=5.6×10⁻⁶ s⁻¹) is approximately twice that reported earlier for acommercial preparation of full length streptavidin (Piran and Riordan,J. Immunol. Meths. 133:141-143 (1990), although monophasic dissociationkinetics were obtained for the recombinant core WT streptavidin asopposed to the multiphasic kinetics observed with commercialpreparations of streptavidin.

                  TABLE III                                                       ______________________________________                                        The rate constant for the dissociation of biotin-                               protein complex (k.sub.off), relative dissociation rate constant             (.increment.k.sub.off), and relative equilibrium dissociation constant       (.increment.K.sub.d)                                                            for WT streptavidin and W79F, W108F, and W120F mutants at 298                 K. .increment.k.sub.off and .increment.K.sub.d are defined relative to      WT streptavidin.                                                                   Protein    k.sub.off (S.sup.-1)                                                                   §.increment.k.sub.off                                                                †.increment.K.sub.d               ______________________________________                                        WT          5.4 × 10.sup.-6                                                                  1             1                                            W79F 3.6 × 10.sup.-5 5.5 ˜10.sup.1                                W108F 9.6 × 10.sup.-5 17 ˜10.sup.1                                W120F 3.7 × 10.sup.-4 70 ˜10.sup.2                              ______________________________________                                         §.increment.k.sub.off = k.sub.off (mutant)/k.sub.off (WT)                †.increment.K.sub.d = K.sub.d (mutant)/K.sub.d (WT)               

The effects of replacing the Trp residues in contact with biotin by Phewere significant and position-dependent. There was a dramatic effect inthe k_(off) upon replacing Trp 120 with Phe: the conservative W120Fmutation resulted in a 70-fold increase in the dissociation rateconstant, relative to WT streptavidin, corresponding to a decrease inthe t_(1/2) from 35 h (WT) to 0.5 h (W120F). The effects of replacingTrp with Phe at positions 79 and 108 were also significant, thoughsmaller in magnitude. The k_(off) for W79F was 5-fold greater, and thatof W108F was 17-fold greater than WT streptavidin. The Δk_(off) of W79Fand W120F relative to WT streptavidin, summarized in Table I, weresimilar in magnitude in their ΔK_(d), suggesting that the decreasedequilibrium affinity of W79F and W120F can be largely accounted for bytheir increased k_(off). W108F, on the other hand, displayed a biotinΔk_(off) that is larger than the ΔK_(d), suggesting that thecorresponding on-rate alterations also exist. These results indicatethat the alterations in the off rate kinetics for W79F and W120F arelargely accounted for by a free energy destabilization of theligand-bound ground state relative to WT streptavidin, whilestabilization of the transition state of W108F relative to WT largelyaccounts for the Δk_(off) of this mutant.

The energetic origins of the marked differences in k_(off) for W79F,W108F, and W120F were further examined by measuring thetemperature-dependence of the dissociation rates, and applyingtransition state theory to determine the enthalpic and entropiccontributions to the activation barrier. The activation thermodynamicparameters, calculated with the assumption of a single transition state,are summarized in Table IV. The exceptionally slow biotin off-rate isdue to a large activation barrier, ΔG_(r).sup.≠ =24.4 kcal mol⁻¹, forbiotin dissociation. The activation barrier is enthalpic in origin,ΔH_(r).sup.≠ =+32 kcal mol⁻¹, with a positive activation entropy of 7.6kcal mol⁻¹ at 298 K. An unexpected finding is that both W79F and W108Fexhibit a larger activation enthalpy of dissociation (+3,+4.5 kcalmol⁻¹) compared to WT streptavidin. However, this Trp->Phe substitutionresults in an even larger positive increase in the activation entropycontribution, as the TΔS_(r) ≠ term for W79F and W108F favorsdissociation by 4-6 kcal mol⁻¹ at 298 K. In direct contrast, the 70-foldfaster off-rate of W120 (corresponding to a ΔΔG_(r) ≠ of -2.1 kcal mol⁻¹relative to WT streptavidin) largely arises from a decreased activationenthalpic barrier (ΔH_(r) ≠=+28.5 kcal mol⁻¹), with an activationentropy that is very similar to WT streptavidin.

                  TABLE IV                                                        ______________________________________                                        Thermodynamic parameters derived from transition                                state analysis of the temperature-dependent dissociation of                   the biotin-streptavidin (WT or mutant) complex.                                        .increment.H.sub.r ≠                                                              .sup.1 T.increment.S.sub.r ≠                                                     .increment.G.sub.r ≠                                                            .sup.2 .increment..increment.G.sub.r                                           ≠                                  Protein (kcal mol.sup.-1) (kal mol.sup.-1) (kal mol.sup.-1) (kal                                                  mol.sup.-1)                             ______________________________________                                        WT     32.0 ± 2.1                                                                            7.6 ± 2.1                                                                            24.4 ± 2.4                                                                         0                                           W79F 34.9 ± 0.6 11.6 ± 0.6 23.3 ± 0.8 -1.1                           W108F 36.5 ± 0.9 13.8 ± 0.9 22.7 ± 1.3 -1.7                          W120F 28.5 ± 1.0  6.6 ± 1.0 21.9 ± 1.4 -2.5                        ______________________________________                                         .sup.1 T = 298K                                                               .sup.2 .increment..increment.G.sub.r ≠ = .increment.G.sub.r             ≠(mutant) - .increment.G.sub.r ≠(WT)                         

The determination of ΔG_(r) ≠ by transition state analysis and theindependent estimation of ΔG⁰ from affinity measurements allows thedrawing of free energy profiles for WT streptavidin and the Trp to Phemutants. Similarly, the enthalpic energy profiles can be drawn from theequilibrium biotin-binding enthalpy (ΔH⁰), independently determined byisothermal titrating calorimetry measurements, and ΔH_(r) ≠ availablefrom transition state analysis of the temperature-dependentprotein-ligand dissociation kinetics. The virtue of this analysis isthat is allows the free energy barriers responsible for the off-rates ofthe different mutants to be delineated in terms of alterations in thetransition state and/or the biotin-bound ground state, and furtherpartitions these free energy changes into enthalpic and entropiccomponents. Upon comparison with the results for WT streptavidin, thethermodynamic effects of mutating specific residues can then be mapped,providing considerable insight into the structure and energetics of thetransition state.

Turning to the free energy profiles for WT streptavidin and the Trp toPhe mutants, the 70-fold decrease in the k_(off) of W120F is believeddue to the destabilization of its biotin-bound ground state relative toWT streptavidin, ΔΔG=+2.7 kcal mol⁻¹ vs. DDG_(r) π=-2.5 kcal mol⁻¹. Thisalteration in the free energy of the ligand-bound state upon mutatingTrp120 to Phe is enthalpically controlled, ΔΔH°=+5.1 kcal mol⁻¹, with afavorable entropy term, TDDS∞=+2.4 kcal mol⁻¹. W79F displays thesmallest decrease in the k_(off), which can also be attributed to thedestabilization of the biotin-bound ground state. The origin of this+0.9 kcal mol⁻¹ ΔΔG° term is different from W120F. The change inequilibrium binding enthalpy actually favors association since ΔΔH°=-1.5kcal mol⁻¹, indicating that the free energy destabilization of thebiotin-bound state caused by mutating Trp70 to Phe is caused by an evenlarger unfavorable entropic alteration, TΔΔS°=-2.4 kcal mol⁻¹.

EXAMPLE III

This Example demonstrates that streptavidin-biotin equilibrium affinityand dissociation kinetics engineered by site-directed mutagenesis allowefficient capture of a biotinylated target molecule while subsequentlyallowing dissociation of the streptavidin-biotinylated target complexunder non-denaturing conditions by exploiting competitive dissociationwith free biotin. The ability to tailor the biotin-binding affinity anddissociation kinetics is used to create chimeric streptavidin tetramerscomposed of subunits with widely differing affinities and biotin offrates.

The Trp120->Ala (W120A) mutant (Example I), which has a K_(a) of 10⁷ M⁻¹and a k_(off) ≦10⁻² s⁻¹ was used to demonstrate the reversibleseparation of a biotinylated molecule. W120A binds to a biotinylatedchromatography support under buffer flow, and can be subsequently elutedwith 2 mM biotin (FIG. 5A). The experiment was performed as follows: 0.4mg of W120A in PBS, pH 7.4 was added to a 1 ml biotin-functionalizedcolumn (Sigma Chemicals, St Louis, Mo.). The column was washed with 5 mlPBS to remove nonspecifically adsorbed protein, followed by elution with2 mM biotin in PBS. All of the added W120A was recovered in the biotinelute as determined by optical absorbance at 280 nm. An identicalexperiment with WT streptavidin resulted in irreversible binding of theprotein to the column which could not be eluted by biotin (FIG. 5B),even after incubation of the column in strong denaturants, which isconsistent with the exceptional stability of biotin-bound streptavidin.The initial binding of W120A and WT streptavidin to an immobilizedbiotin column is a consequence of their high affinity. Upon subsequentaddition of biotin to the buffer flow, the fast off rate of theW120A-biotin complex allows effective competition of the free biotin forthe immobilized (column bound) biotin, leading to displacement of boundW120A. In the case of WT streptavidin, the extremely slow biotin offrate (t_(1/2) =35 h) precludes the dissociation of the immobilizedbiotin-WT streptavidin, resulting in essentially irreversible binding ofthe protein to the column.

The fast off-rate kinetics of W120A also permitted regeneration ofligand-free protein by ultrafiltration or dialysis. The reverseexperiment, the binding of biotinylated target to immobilized W120A, andsubsequent elution with free biotin is equivalent to this experiment.The binding of W120A to immobilized biotin, and its elution with freebiotin, and the irreversible binding of WT streptavidin, highlights theimportance of controlling both the affinity and off-rate kinetics in theoptimization of affinity separations.

Chimeric tetramers with mixed WT streptavidin and W120A subunits werecreated by mixing equimolar amounts of WT streptavidin and W120A,denaturation in guanidine thiocyanate, followed by slow renaturation ofthe denatured protein by dialysis, as follows: 2.4 mg each of WTstreptavidin and W120A (protein volume=1.13 ml) were incubated in 7.6ml, 6M guanidine thiocyanate for 1 h at room temp, and dialyzedovernight in 4 L 25 mM Tris.Cl, 80 mM glycine at 4° C. The dialysate wasthen recovered for further purification. Upon refolding, a number ofdistinct protein populations result: chimeric tetramers with subunitstoichiometries of 1:3. 2:2 and 3:1 (WT:W120A), parent WT and W120A, anddenatured protein subunits caused by aggregation/misfolding. Theseparation of the chimeric tetramers from parent W120A tetramers andmisfolded/aggregated protein utilized 2-iminobiotin affinitychromatography. W120A exhibits <10³ M⁻¹ affinity for 2-iminobiotin.Passage of the dialysate over an iminobiotin column, and elution ofbound protein at pH 4, resulted in the recovery of .sup.˜ 20% of theoriginal protein, consisting of chimeric tetramers and parent WTstreptavidin (FIG. 6A). The dialysate was equilibrated in binding buffer(50 mM sodium carbonate, 0.5 M NaCl, pH 11) and passed over a 10 mliminobiotin column (Pierce, Rockford, Ill.). Unbound protein (.sup.˜ 4mg) was eluted in binding buffer, and specifically bound protein (0.8mg) was eluted in 50 mM sodium acetate, 0.5 M NaCl, pH 4. The recoveredprotein was concentrated, and exchanged into PBS, pH 7.4 bydiafiltration. The separation of chimeric tetramers from parent WTstreptavidin used the vastly differing biotin off rates of WTstreptavidin and W120A. The protein was incubated with .sup.˜ 2-foldexcess of biotin, and exhaustively ultrafiltered to remove the excessbiotin. Since the t_(1/2) of WT-biotin complex is 35 h, while that ofW120A is <10² s, this results in the removal of biotin from asubstantial fraction of W120A biotin-binding sites as well. The proteinwas then passed over a biotin column, washed with buffer, and theneluted with 2 mM biotin (FIG. 6B). Parent WT streptavidin tetramerssaturated with biotin did not bind to the column, while WT streptavidintetramers with free biotin-binding sites bind irreversibly to the biotincolumn (cf. FIG. 5B). This step effectively separates the chimerictetramers from the parent WT streptavidin tetramers.

Protein-containing fractions eluted with biotin migrated similarly tobiotin-bound streptavidin tetramers in native polyacrylamide gelelectrophoresis, indicating similar quaternary structure (20 μl of themixed tetramers at μM concentration were analyzed on a 8% acrylamide gelin the absence of SDS on a Miniprotean gel electrophoresis apparatus(BioRad Inc., Hercules, Calif.)). Direct corroboration that the proteinpopulation was composed of chimeric tetramers was obtained byelectrospray mass spectrometry (ESMS). The chimeric streptavidintetramers were analyzed by ESMS under conditions that favor preservationof noncovalent association. Protein concentrations (WT+biotin, W120A andmixed tetramers) were 10-50 μM tetramer in 10 mM ammonium acetate, pH8.6. Experiments to detect the tetrameric charge states were conductedon a low frequency extended mass range single quadrupole massspectrometer (Extrel Corp.). Instrument details and general operatingconditions can be found in Light-Wahl et al., J. Amer Chem. Soc.115:5869 (1993); Light-Wahl et al., J. Amer Chem. Soc. 116: 5271 (1994);Schwartz et al., J. Amer.Soc. Mass Spectrom. 5:201-204 (1994); andSchwartz et al., J. Amer. Soc. Mass Spectrom., in press (1995),incorporated herein by reference. The results are shown in Table V.

                  TABLE V                                                         ______________________________________                                        ESMS results of protein eluted by buffer and biotin                             in biotin chromatography of mixed tetramers. Biotin eluted                    protein corresponds to FIG. 6B, while buffer eluted protein                   corresponds to FIG. 6B.                                                                  Charge  Centroid                                                                             FWHM  Intensity                                                                            Mass                                   Sample State (m/z) (m/z) (counts) (Da)                                      ______________________________________                                        Biotin eluted                                                                          +15     3555     95    2376   53353 ± 43                            protein +14 3815 110  4123                                                    Buffer eluted +15 3580 90 3586 53751 ± 66                                  protein +14 3845 90 2920                                                      WT tetramers +15 3540 75 4897 53136 ± 51                                    +14 3800 60 3808                                                           ______________________________________                                    

Both the shifted peak position of the +14 and +15 charge state(indicative of tetrameric streptavidin) of the putative chimerictetramer, and its greater full width at half maximum (FWHM) incomparison with the position and FWHM of parent WT+biotin or W120Atetramers (Table VI) is consistent with the presence of chimerictetramers comprising biotin-bound WT and biotin-free W120A subunits. Thealternative explanation, that the shift in position of the +14 chargestate and broader FWHM could arise from a mixed population ofbiotin-bound WT and W120A tetramers is precluded by the affinitypurification employed.

                  TABLE VI                                                        ______________________________________                                        Calculated m/z values for tetrameric charge states                              for different stoichiometric association of W120A subunits and                WT + biotin subunits.                                                         Calculated m/z                                                                      Sample        +15       +14  Mass (Da)                                ______________________________________                                        W120A         3510        3760   52624                                          W120A:WT + biotin 3535 3785 52983                                             (3:1)                                                                         W120A:WT + biotin 3555 3810 53342                                             (2:2)                                                                         W120A:WT + biotin 3580 3835 53701                                             WT + biotin 3605 3860 54060                                                   WT 3540 3795 53084                                                          ______________________________________                                    

Further corroboration that both WT subunits and W120A subunits werepresent in this protein was obtained by analyzing the protein underconditions that favored dissociation of the tetramer into monomers. TheESMS experiment to detect monomeric ions was conducted on a FinniganMATtriple quadrupole mass spectrometer under capillary heating conditionsthat lead to dissociation of the tetramer into +6 and +7 monomeric ions(capillary voltage=+2.7 kV, capillary temperature=180° C.); samplepreparation was as described above. These ESMS results indicated thatthe peaks associated with the +7 and +6 charge state arose from both WTand W120A subunits.

The subunit mixing of two distinct populations of a multisubunit proteinwas demonstrated by resonance energy transfer experiments where onepopulation was labeled with a fluorescence energy donor and the otherwith a complementary fluorescence energy acceptor, as generallydescribed in Erijman and Weber, Biochemistry 30: 1595-1599 (1991), andErijman and Weber, Photochem. Photobiol. 57: 411-415 (1993),incorporated herein by reference. Equimolar amounts of fluoresceinisothiocyanate-labeled WT streptavidin (FITC-WT streptavidin) and7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin-labeled WTstreptavidin (CPI-WT streptavidin) were mixed, denatured in 6 Mguanidine isothiocyanate, and allowed to renature by dialysis in buffer,as follows: WT streptavidin was labeled with FITC or CPI (MolecularProbes, Eugene, Oreg.) by incubating protein in a ten-fold molar excessof FITC or CPI in 50 mM Na₂ CO₃, 150 mM NaCl, pH 9.0, for 2 h at roomtemp. The labeled proteins were then separated from unbound fluorophoreby gel filtration (PD-10, Pharmacia, Piscataway, N.J.). Fluorophorelabeling ratios were typically 3-4 per tetramer as determined by opticalabsorbance measurements. Equimolar mixtures of FITC-labeled WTstreptavidin and CPI-labeled WT streptavidin were incubated in 1.5 ml 6Mguanidine thiocyanate for 1 h at room temp, and then dialyzed against 25mM Tris.Cl, 80 mM glycine, pH 8.6. Fluorescence measurements wereperformed on a Hitachi F-5000 spectrofluorimeter: excitation=385 nm,emission=400-500 nm. While care was taken to ensure identicalconcentration of the renatured protein tetramers and the control,dilution effects arising from dialysis were corrected for bynormalization to the tryptophan fluorescence of each sample.

Thus, upon physical mixing of these two distinct tetrameric populations,followed by chaotrope-induced denaturation and protein refolding, theoccurrence of resonance energy transfer clearly demonstrated thecreation of chimeric tetramers, consisting of two different subunittypes. The results with WT streptavidin are shown in FIG. 1: Theemission spectrum of the renatured mixture displayed a significantenhancement in the intensity of FITC-labeled WT streptavidin, thefluorescence energy acceptor, compared to a mixture of these two labeledproteins that had not undergone denaturation/renaturation, indicatingthe occurrence of fluorescence energy transfer. The observation offluorescence resonance energy transfer is diagnostic of the presence ofdonor and acceptor labels on the same (tetrameric) molecule, aconsequence of the creation of chimeric streptavidin tetramersconsisting of subunits of both FITC-labeled WT streptavidin andCPI-labeled WT streptavidin.

These results demonstrate that a targeting component (biotin bound to WTsubunits) and an imaging component (binding of the chimeric streptavidintetramer to a biotin column by W120A subunits) can be separated by theuse of chimeric streptavidin tetramers with different subunit affinitiesand biotin-dissociation kinetics. Thus, for drug delivery, imaging, andother such uses, the chimeric tetramer is loaded with a biotinylatedmolecule (e.g., biotinylated antibody) such that it is selectivelypartitioned to the high affinity subunits by virtue of the fast off-rateof the lower affinity subunits. The biotinylated target/chimera complexis targeted to the desired site via the antibody. The imaging agent ordrug is circulated and captured by the lower affinity subunits.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 11                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - TTTCGCAGCA ACGGTCCAAC CCAGAGCGGT TCCAGAA      - #                      - #      37                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TTTGAAAGCA ACGGTCCAAC CCAGAGCGGT TCCAGA      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - ACCGCGTCTG GTCAGTACGT TGGTGGTGCT GAA       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CCTTCTCTGG TCAGTACGTT GGTGGTGCTG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GGTCGTACCG GAGGTCAGCA GCGACTGG         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GGTCGTACCG GAGGTCAGCA GGACCTGG         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AGTTCTTGTT GACCTCCGGC ACCACCGAAG CTAACGCTTG G    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - CGCGGCTAAA TCCACCCTGG TTGGTCACGA CACCTTCACC AAAGTTAA  - #                    48                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CGCGTTTAAA TCCACCCTGG TTGGTCACGA CACCTTCACC AAAGTTAA  - #                    48                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - AGCTTTTATT AGGAAGCTGC AGACGGTTTA ACTTTGGTGA AGGTGTCGTG AC -             #CAACCAGG     60                                                                 - - GTGGATTTAG C               - #                  - #                      - #       71                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AGCTTTTATT AGGAAGCTGC AGACGGTTTA ACTTTGGTGA AGGTGTCGTG AC -             #CAACCAGG     60                                                                 - - GTGGATTTAA A               - #                  - #                      - #       71                                                                __________________________________________________________________________

What is claimed is:
 1. An isolated chimeric streptavidin tetramercomprising between one and three modified monomers,wherein the tetrameris formed by mixing wildtype monomers and a single type of modifiedmonomer, wherein the chimeric tetramer has one or more of the followingcharacteristics:(i) an overall altered binding affinity for biotin or acompound comprising biotin wherein a subunit modification consists ofeither a substitution of a hydrophobic amino acid for one or more aminoacids present in the biotin binding domain or a deletion of one or moreamino acids in the biotin binding domain, (ii) an altered subunitaffinity conferring an enhanced sensitivity to hydrostatic pressurewherein a subunit modification consists of the substitution of an aminoacid selected from the group consisting of a positively-charged aminoacid, a negatively charged amino acid, or a cysteine for an amino acidpresent either in the region of a monomer-monomer interface or in theregion of the dimer-dimer interface, and (iii) a molecular labelpermitting monitoring of subunit association into tetramers havingspecific stoichiometric ratios of modified and unmodified subunitswherein a subunit modification consists of the substitution of cysteinefor asparagine at amino acid position 94 and covalent attachment of thelabel to the cysteine, and, (iv) a capacity to bind to a molecule otherthan streptavidin, biotin, or a compound comprising biotin, wherein asubunit modification consists of the substitution of cysteine forasparagine at amino acid position 94 and covalent attachment of themolecule to the cysteine.
 2. The chimeric streptavidin tetramer of claim1, wherein the streptavidin tetramer has an overall altered bindingaffinity for biotin or a compound comprising biotin wherein a subunitmodification consists of either a substitution of a hydrophobic aminoacid for one or more amino acids present in the biotin binding domain ora deletion of one or more amino acids in the biotin binding domain. 3.The chimeric streptavidin tetramer of claim 1, wherein the modifiedmonomer further comprises a label, linker, drug, toxin, targetingmolecule, metal, or enzyme.
 4. The chimeric streptavidin tetramer ofclaim 1, wherein the subunit modification consists of the substitutionof a cysteine for an amino acid present either in the region of amonomer-monomer interface or in the region of the dimer-dimer interface.5. The chimeric streptavidin tetramer of claim 4, wherein the cysteineis situated at position H127.
 6. The chimeric streptavidin tetramer ofclaim 1, wherein the streptavidin tetramer has an overall reducedbinding affinity for biotin or a compound comprising biotin wherein asubunit modification consists of either a substitution of a hydrophobicamino acid for one or more amino acids present in the biotin bindingdomain or a deletion of one or more amino acids in the biotin bindingdomain.
 7. The chimeric streptavidin tetramer of claim 1, wherein thestreptavidin tetramer has a capacity to bind to a molecule other thanstreptavidin, biotin, or a compound comprising biotin, wherein a subunitmodification consists of the substitution of cysteine for asparagine atamino acid position 94 and covalent attachment of the molecule to thecysteine.
 8. The chimeric streptavidin tetramer of claim 1, wherein thestreptavidin tetramer has an altered subunit affinity conferring anenhanced sensitivity to hydrostatic pressure wherein a subunitmodification consists of the substitution of an amino acid selected fromthe group consisting of a positively-charged amino acid, a negativelycharged amino acid, or a cysteine for an amino acid present either inthe region of a monomer-monomer interface or in the region of thedimer-dimer interface.
 9. A streptavidin tetramer having an alteredsubunit affinity conferring an enhanced sensitivity to hydrostaticpressure wherein a subunit modification consists of the substitution ofan amino acid selected from the group consisting of a positively-chargedamino acids a negatively charged amino acid, or a cysteine for an aminoacid present either in the region of a monomer-monomer interface or inthe region of the dimer-dimer interface.
 10. The chimeric streptavidintetramer of claim 9, wherein the subunit modification consists of thesubstitution of a cysteine for an amino acid present either in theregion of a monomer-monomer interface or in the region of thedimer-dimer interface.
 11. The chimeric streptavidin tetramer of claim10, wherein the cysteine is situated at position H127.
 12. A chimericstreptavidin tetramer comprising between one and three modifiedmonomers,wherein the chimeric tetramer has one or more of the followingcharacteristics:(i) an overall altered binding affinity for biotin or acompound comprising biotin wherein a subunit modification consists ofeither a substitution of a hydrophobic amino acid for one or more aminoacids present in the biotin binding domain or a deletion of one or moreamino acids in the biotin binding domain, (ii) an altered subunitaffinity conferring an enhanced sensitivity to hydrostatic pressurewherein a subunit modification consists of the substitution of an aminoacid selected from the group consisting of a positively-charged aminoacid, a negatively charged amino acid, or a cysteine for an amino acidpresent either in the region of a monomer-monomer interface or in theregion of the dimer-dimer interface, and (iii) a molecular labelpermitting monitoring of subunit association into tetramers havingspecific stoichiometric ratios of modified and unmodified subunitswherein a subunit modification consists of the substitution of cysteinefor asparagine at amino acid position 94 and covalent attachment of thelabel to the cysteine, and, (iv) a capacity to bind to a molecule otherthan streptavidin, biotin, or a compound comprising biotin, wherein asubunit modification consists of the substitution of cysteine forasparagine at amino acid position 94 and covalent attachment of themolecule to the cysteine.
 13. The chimeric tetramer of claim 12 whereinthe chimeric tetramer has one or more of the followingcharacteristics:(i) an overall altered binding affinity for biotin or acompound comprising biotin wherein a subunit modification consists ofeither a substitution of a hydrophobic amino acid for one or more aminoacids present in the biotin binding domain or a deletion of one or moreamino acids in the biotin binding domain, and (ii) an altered subunitaffinity conferring an enhanced sensitivity to hydrostatic pressurewherein a subunit modification consists of the substitution of an aminoacid selected from the group consisting of a positively-charged aminoacid, a negatively charged amino acid, or a cysteine for an amino acidpresent either in the region of a monomer-monomer interface or in theregion of the dimer-dimer interface.
 14. An isolated collection ofchimeric streptavidin tetramers wherein the tetramers are formed bymixing wildtype monomers and a single type of modified monomer,whereinthe chimeric tetramer has one or more of the followingcharacteristics:(i) an overall altered binding affinity for biotin or acompound comprising biotin wherein a subunit modification consists ofeither a substitution of a hydrophobic amino acid for one or more aminoacids present in the biotin binding domain or a deletion of one or moreamino acids in the biotin binding domain, (ii) an altered subunitaffinity conferring an enhanced sensitivity to hydrostatic pressurewherein a subunit modification consists of the substitution of an aminoacid selected from the group consisting of a positively-charged aminoacid, a negatively charged amino acid or a cysteine for an amino acidpresent either in the region of a monomer-monomer interface or in theregion of the dimer-dimer interface, and (iii) a molecular labelpermitting monitoring of subunit association into tetramers havingspecific stoichiometric ratios of modified and unmodified subunitswherein a subunit modification consists of the substitution of cysteinefor asparagine at amino acid position 94 and covalent attachment of thelabel to the cysteine, and, (iv) a capacity to bind to a molecule otherthan streptavidin, biotin, or a compound comprising biotin, wherein asubunit modification consists of the substitution of cysteine forasparagine at amino acid position 94 and covalent attachment of themolecule to the cysteine.
 15. An isolated chimeric streptavidin tetramercomprising between one and three modified monomers,wherein the tetrameris formed by mixing wildtype monomers and one, two, or three types ofmodified monomer, wherein the chimeric tetramer has one or more of thefollowing characteristics:(i) an overall altered binding affinity forbiotin or a compound comprising biotin wherein a subunit modificationconsists of either a substitution of a hydrophobic amino acid for one ormore amino acids present in the biotin binding domain or a deletion ofone or more amino acids in the biotin binding domain, (ii) an alteredsubunit affinity conferring an enhanced sensitivity to hydrostaticpressure wherein a subunit modification consists of the substitution ofan amino acid selected from the group consisting of a positively-chargedamino acid, a negatively charged amino acid, or a cysteine for an aminoacid present either in the region of a monomer-monomer interface or inthe region of the dimer-dimer interface, and (iii) a molecular labelpermitting monitoring of subunit association into tetramers havingspecific stoichiometric ratios of modified and unmodified subunitswherein a subunit modification consists of the substitution of cysteinefor asparagine at amino acid position 94 and covalent attachment of thelabel to the cysteine, and, (iv) a capacity to bind to a molecule otherthan streptavidin, biotin, or a compound comprising biotin, wherein asubunit modification consists of the substitution of cysteine forasparagine at amino acid position 94 and covalent attachment of themolecule to the cysteine.